Immune tolerance is central to the immune system's ability to differentiate between self and foreign proteins. Central tolerance is initially achieved during thymic selection by the deletion of self-reactive T cells. However, central tolerance is incomplete, and further immune regulation is required in the periphery. Peripheral mechanisms of T cell regulation include the induction of anergy, activation induced cell death, and modulation of T cell activity.
Regulatory T cells are fundamental in controlling various immune responses. Absence or defective function of regulatory T cells has been correlated with autoimmunity in humans, whereas their presence has been associated with tolerance. Compelling data from preclinical animal models indicates that adoptive transfer of regulatory T cells can prevent or cure several T cell-mediated diseases, including autoimmune diseases and allograft rejection by restoring immune tolerance to self antigens or alloantigens. Three categories of regulatory T cells have been described within the CD4+ T lymphocyte cell population: TH3 cells, Type 1 regulatory cells, and CD4+CD25+ T regulatory cells. TH3 cells function via the secretion of TGF-β and can be generated in vitro by stimulation in the presence of IL-4 or in vivo through oral administration of low dose antigens (Chen et al., Science 265:1237-1240, 1994; Inobe et al., Eur. J. Immunol. 28:2780-2790, 1998). Type 1 regulatory T cells suppress T cells through the production of IL-10 and TGF-β and are derived by stimulation of memory T cells in the presence of IL-10 (Groux et al., Nature 389:737-742, 1996; Groux et al., J. Exp. Med. 184:19-29, 1996). CD4+CD25+ regulatory T cells are thought to function as a regulator of autoimmunity by suppressing the proliferation and/or cytokine production of CD4+CD25− T cell responder cells at the site of inflammation. Furthermore, these T regulatory cells decrease the magnitude of the immune response, allowing innocuous antigen to be removed without inducing pathology.
CD4+CD25+ regulatory T cells are present in both humans and mice and are characterized by expression of CD25 (for review, see Sakaguchi et al., Immunol. Rev. 182:18-32). Regulatory T cells isolated from human peripheral blood are highly differentiated memory cells based on their FACS staining characteristics and short telomere length and historically are thought to be derived from the thymus (Taams et al., Eur. J. Immunol. 32:1621-1630, 2002; Jonuleit et al., J. Exp. Med. 193:1285-1294, 2001). In humans, regulatory T cells are believed to represent 1-3% of all CD4+ T cells and require activation to induce suppressor function. The suppressive function of these regulatory T cells is mainly mediated via cell-cell contact and is abrogated by the addition of IL-2 (Baecher-Allan et al., J. Immunol 167:1245-1253, 2001).
The regulatory T cell population is reduced in autoimmune-prone animals and humans (see Salomon et al., Immunity 12:431-440, 2000; Kukreja et al., J. Clin. Invest. 109:131-140, 2002). Mice carrying the X-linked scurfy mutation develop a multi-organ autoimmune disease and lack conventional CD4+CD25+ regulatory T cells (Fontenot et al., Nat. Immunol. 4:330-336, 2003; Khattri et al., Nat. Immunol. 4:337-342, 2003). It has been shown that the gene mutated in these mice is FoxP3, which encodes a member of the forkhead/winged helix family and acts as a transcriptional repressor (Schubert et al., J. Biol. Chem. 276:37672-37679, 2001). In mice, FoxP3 has been shown to be expressed exclusively in CD4+CD25+ regulatory T cells and is not induced upon activation of CD25− cells. However, when FoxP3 is introduced via retrovirus or via transgene expression, naive CD4+CD25− T cells are converted to regulatory T cells (Hori et al., Science 299:1057-1061, 2003). In humans, it has been noted that mutations in FoxP3 lead to a severe lymphoproliferative disorder known as IPEX (immunodysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome, characterized by lymphoproliferative disease, insulin-dependent diabetes, thyroiditis, eczema and death at an early age (see Wildin et al., J. Med. Genet. 39:537-545, 2002).
The CD4+CD25+ regulatory population is heterogeneous, as 20-30% also express HLA-DR. The DR+ regulatory T cells inhibit T-cell proliferation and cytokine production via an early contact-dependent mechanism that is associated with an additional induction of FoxP3 mRNA. In contrast, DR− regulatory T cells do not induce early contact-dependant suppression but rather initially enhance secretion of IL-10 and IL-4. Eventually, DR− regulatory T cells induce a late suppression of proliferation that is associated with a delayed increase in FoxP3 mRNA by the regulatory T cells. Thus, both DR+ and DR− regulatory T cells can suppress via a cell-contact-mediated mechanism, but the DR− population can also suppress by inducing the secretion of IL-10. Therefore, it is possible that different types of autoimmune diseases may be associated with a defect in suppression by either DR+ or DR− regulatory T cells.
Due to their low frequency in peripheral blood, freshly isolated human CD4+CD25+ T cells with suppressive function are difficult to isolate and expand. In the autoimmune NOD mouse model, one group of investigators has recently isolated naturally occurring antigen-specific regulatory T cells from mouse spleen and lymph nodes. These regulatory T cells were expanded ex vivo and transferred to the diabetic prone NOD mouse. Transplantation of these regulatory T cells was demonstrated to suppress the development of diabetes (Tang et al., J. Exp. Med. 199:1455-1465, 2004, Masteller et al., J. Immunol 175:3053-3059, 2005; Tarbell et al., J. Exp Med 199:1467-1477, 2004). This approach demonstrates the therapeutic benefit of regulatory T cell transfer to treat autoimmune disease. However, the approach used in the NOD mouse model is not therapeutically applicable to human subjects, due to the requirement that a large number of rare CD4+CD25+ T cells (approximately 4% of circulating T cells) need to be isolated from the peripheral blood. Further, this mouse model contains a single fixed T cell receptor (TCR) and does not address the problem of following TCR repertoire evolution or identifying antigen-specific T cells in complex systems where a polyclonal T cell response is present. Similar studies have not been possible in human subjects due to the low frequency of antigen-specific regulatory T cells circulating in the peripheral blood, especially with respect to autoreactive T cells.
Type I regulatory cells arise in the periphery after encounter with antigen in the presence of IL-10. The unique cytokine production profile (IL-2low/− IL-4−, IL-5+, IL-10+, TGF-β+) distinguishes Type I regulatory cells from T helper 0 (T01) and TH2 cells. To date, no specific cell-surface markers for Type I regulatory cells have been identified. Type I regulatory cells have a very low proliferative capacity following activation in vitro through the T cell receptor, in part due to autocrine production of IL-10. Type I regulatory cells regulate immune responses through the secretion of the immunosuppressive cytokines IL-10 and TGF-β, and they suppress both naïve and memory T cell responses and downregulate the expression of co-stimulatory molecules and pro-inflammatory cytokines by antigen-presenting cells. Furthermore, Type I regulatory cells favor the production of IgD, IgA, and IgG by B cells. Importantly, Type I regulatory cells are inducible, antigen-specific, and need to be activated through their TCR to exert their suppressive functions. However, once activated, they mediate suppression in an antigen non-specific manner (Roncarolo et al. Immunol. Rev. 2006. 212: 28-50).
Given the important role regulatory T cells play in immune tolerance, there is a need to develop methods for generating, selecting and expanding human regulatory T cells for use in the treatment and/or prevention of autoimmune diseases, inflammatory conditions, and for the prevention of graft rejection in a recipient following solid organ or stem cell transplantation.